Unconventional myosins comprise an important class of molecular motor proteins. They are involved in a myriad of different functions in eukaryotes, including transport of vesicles and mRNAs, actin reorganization, and predominantly structural functions. Several myosins (Ic, VI, VIla, and XV) have essential roles in hair-cell function. To ascertain the specific cellular roles of these respective motors in hair cells, a chemical-genetic approach (already validated for myosin-1c in slow adaptation) will be utilized. Mutant myosins will be engineered to enlarge their ATP binding pockets, which will sensitize them to synthetic ADP analogs. Knock-in or transgenic mice expressing these sensitized motors will allow the study of each motor's hypothesized role(s) in slow adaptation, actin bundle development, or apical endocytosis. This approach provides several advantages; foremost of which is tight temporal control over the specific, selective inhibition of myosin function. The ability to selectively 'lock down' a specific myosin onto actin filaments (rigor complex) enables a more rigorous determination of in vivo function without the artifacts caused by overexpression. [unreadable] [unreadable]